An important but poorly understood aspect of mammalian follicle development involves the regulation of theca cell proliferation. To investigate the premise that growth factors regulate theca cell proliferation, porcine theca cells were prepared by collagenase/DN'ase digestion of follicle linings after the removal of the granulosa cells and allowed to attach for 24 h. This method provided a monolayer of theca cells that had little if any granulosa cell contamination and which secreted high levels of androstenedione relative to granulosa cells during moderate-term culture (33-fold difference, P less than 0.01). In medium containing fetal calf serum (10%), theca cells were significantly more responsive to platelet-derived growth factor (PDGF) than epidermal growth factor (EGF) in terms of proliferation (13.4 +/- 0.2-vs. 7.0 +/- 0.1-fold increases relative to the initial cell count, P less than 0.05). This is in contrast to granulosa cells which were significantly more responsive to EGF than PDGF (7.1 +/- 0.1 vs. 4.0 +/- 0.2 fold-increases, P less than 0.05). Since serum has been shown to contain both EGF and PDGF, proliferation studies were performed using plasma-derived serum (PDS) which is growth factor restricted to examine more closely the direct effects of growth factors. In medium containing 0.25% PDS and within experiments, PDGF (1-25 ng/ml) stimulated theca cell proliferation in a dose-dependent manner (2.3-fold increase relative to controls, P less than 0.05) whereas EGF did not. EGF, however, markedly enhanced the proliferative action of PDGF (6.4-fold increase relative to controls, P less than 0.05). Insulin-like growth factor I and low density lipoprotein, factors which enhance markedly the proliferative effects of EGF and PDGF in terms of granulosa cell proliferation, exhibited only a modest synergistic effect with respect to EGF and PDGF upon theca cells (9.5-fold increase vs. a 6.4-fold increase above controls, P less than 0.05). Temporal studies in vitro indicate that theca cell proliferation is low during the first 3-day exposure to growth factors irrespective of treatment (a 2-fold increase over the seeding density). During the second 3-day exposure, however, theca cell proliferation increases 4- to 5-fold. The temporal pattern of theca cell proliferation stimulated by fetal calf serum supplemented with EGF or PDGF and PDS-containing medium supplemented with PDGF, EGF, insulin-like growth factor I, and low density lipoprotein is similar. These results suggest that PDGF is a major mitogen toward porcine theca cells and that EGF greatly enhances its activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Recent studies suggest that epidermal growth factor (EGF) and/or transforming growth factor-alpha (TGF-alpha) and insulin-like growth factor-I (IGF-I) act synergistically to promote granulosa cell proliferation in vitro suggesting a similar role in vivo. Using a serum-restricted, monolayer culture system containing very low levels of platelet-poor plasma-derived serum (PPPDS), the facilitative roles of platelet-derived growth factor (PDGF) and low density lipoprotein (LDL) with respect to growth factor-stimulated granulosa cell proliferation were investigated. In nutrient medium containing only 0.1% PPPDS, PDGF (1-25 ng/ml) had no effect upon granulosa cell proliferation. When combined with EGF, which alone does not stimulate granulosa cell proliferation, PDGF dose-dependently increased cell proliferation to levels obtained with 10% fetal calf serum (2.4-fold increase relative to controls, P less than 0.05). When combined with EGF and IGF-I, a combination which does stimulate mitosis in granulosa cells, PDGF again dose-dependently enhanced proliferation (P less than 0.05). The extent of proliferation obtained with EGF + IGF-I + PDGF was consistently greater than that obtained with 10% fetal calf serum (P less than 0.05) but significantly less than that obtained with EGF + fetal calf serum, a treatment which stimulates rapid granulosa cell proliferation. LDL has been shown to greatly enhance granulosa cell steroidogenesis by providing exogenous cholesterol. However, cholesterol is also required for plasma membrane biosynthesis and cell growth. LDL alone, had no effect upon porcine granulosa cell proliferation relative to media controls (0.1% PPPDS) nor did it synergize with any single growth factor to induce mitosis. When combined with EGF + IGF-I, and EGF + PDGF, but not PDGF + IGF-I, LDL dose-dependently (1-25 micrograms/ml) enhanced proliferation (P less than 0.05) to levels equivalent to that obtained with 10% fetal calf serum. When combined with EGF, IGF-I, and PDGF, LDL at 10 micrograms/ml enhanced proliferation to an extent equivalent with EGF + fetal calf serum (a 5.4-fold increase relative to media controls). High density lipoprotein did not itself stimulate proliferation nor did it facilitate proliferation mediated by growth factors. When maintained in medium alone (0.1% PPPDS), the cell population doubling time was 8.0 +/- 0.5 days. In the presence of EGF, IGF-I, PDGF, and LDL (10, 10, and 5 ng/ml, and 10 micrograms/ml, respectively) the doubling time was reduced to 2.0 +/- 0.1 days.(ABSTRACT TRUNCATED AT 400 WORDS)